Our long-term goal is to help develop new molecularly targeted colon cancer treatments. Angiogenesis is critical for colonic tumorigenesis. The peroxisome proliferator-activated receptor-delta (PPAR-d) is upregulated in human colon cancer. Published data are discordant on the effects of germ line PPAR-d genetic knock out (KO) on intestinal tumorigenesis in APCMin mice and the effects of PPAR-d agonist on vascular endothelial growth factor (VEGF) expression in cancer cell lines and intestinal adenomas of APCMin mice. The impact of PPAR-d upregulation in cancer cells on angiogenesis and tumorigenesis remains to be determined. Our preliminary data show that targeted intestinal PPAR-d KO profoundly inhibited azoxymethane-induced murine colonic tumorigenesis and VEGF expression; PPAR-d re-expression in PPAR-d null HCT-116 (PPAR-d-KO) cells restored their ability to form liver metastases and enhanced VEGF and interleukin-8 (IL-8) expression; and liposomal PPAR-d siRNA inhibited tumorigenesis and PPAR-d, VEGF, and IL-8 expression in HCT-116 in mice. We hypothesize that PPAR-d overexpression in colon cancer cells upregulates VEGF and IL-8 expression to promote tumor angiogenesis and tumorigenesis. Aim 1 is to determine if PPAR-d specific expression in colon cancer cells promotes tumorigenesis and angiogenesis and upregulates VEGF and IL-8 expression, by examining the effects of PPAR-d re-expression in PPAR-d-KO cells on angiogenesis markers (e.g. CD31 immunohistochemistry; FITC-lectin assay), tumorigenesis (liver and lung metastasis formation), and VEGF and IL-8 expression (mRNA by quantitative RT-PCR, protein by ELISA) in nude mice. Aim 2 is to determine whether PPAR-d knockdown via systemic delivery of liposomal PPAR-d siRNA to colon cancer cells in vivo is sufficient to inhibit tumorigenesis and angiogenesis and downregulate VEGF and IL-8 expression, by examining the effects of liposomal PPAR-d siRNA on angiogenesis, tumorigenesis (liver and lung metastasis formation by HCT-116 and HT-29 cells in nude mice), and PPAR-d, VEGF, and IL-8 expression. Aim 3 is to determine VEGF role in PPAR-d promotion of angiogenesis and tumorigenesis, by examining in nude mice the effects of VEGF overexpression in PPAR-d-KO cells on angiogenesis and tumorigenesis; assessing the effects of VEGF liposomal-siRNA knockdown on angiogenesis and tumorigenesis promoted by PPAR-d re-expression in PPAR-d-KO cells; and comparing the effects of PPAR-d and VEGF downregulation (via liposomal siRNA) on angiogenesis and liver and lung metastasis formation by HCT-116 and HT-29 cells. Aim 4 is to determine IL-8 role in PPAR-d promotion of angiogenesis and tumorigenesis, as done for VEGF in specific Aim 3. In Aims 3 and 4, we will also investigate if PPAR-d binds to the VEGF and IL-8 promoters to enhance their transcription, using VEGF and IL-8 promoter-luciferase deletion construct assays, EMSA, and ChIP/real-time PCR in HCT- 116 cells with wild-type PPAR-d expression, PPAR-d overexpression, or PPAR-d-KO. Confirmation of the tested hypothesis could lead to developing PPAR-d-targeted therapy to inhibit tumorigenesis. PUBLIC HEALTH RELEVANCE: Increased production of the protein peroxisome proliferator-activated receptor delta (PPAR-d) is associated with colon cancer development; however, the molecular mechanisms involved need to be clarified. The proposed project aims to improve understanding of the molecular mechanisms by which PPAR-d contributes to aspects of colon cancer development, especially the ability of tumors to make new blood vessels. This improved understanding is expected to help determine whether PPAR-d would be a suitable molecular target for development of new drugs to treat colon cancer.